Assessment of various detection methods of biofilm formation in Staphylococcus aureus clinical isolates

Document Type : Original Article

Authors

Department of Microbiology, Islamic Azad University, Qom Branch, Qom, Iran

Abstract

Background and Objective: As an important factor in pathogenicity, the ability of Staphylococcus aureus to produce biofilm increases its stability in the environment and living host. Biofilms formed by S.aureus, especially those related to the implant of medical devices, can act as a physical barrier against antibiotics and the host's immune system, leading to chronic or persistent infections. Hence, implementation of efficient diagnostic tests for the detection of biofilm formation can help reduce the disease burden. The purpose of this study was to assess different methods for the detection of biofilm formation in 40 S.aureus isolates.
Materials and Methods: A total of 40 non-duplicate S. aureus clinical isolates were identified. Biofilm formation was detected by Tissue Culture Plate (TCP), tube method (TM) and Congo red agar (CRA) methods and PCR assay was used to detect icaA and icaD genes.
 
Results: Among all S. aureus isolates, 21(52.5%) contained both icaA and icaD genes and icaD gene was present in all biofilm positive isolates. Tissue culture plate, Congo red agar, and tube method detected 30%, 42.5%, and 67.5% biofilm formation isolates, respectively.
Conclusion: According to the results, tissue culture plate with supplemented glucose showed the best correlation with the results of molecular assay and can be used as a reliable method to detect biofilm formation in clinical isolates of S. aureus.

Keywords


  1. Kouidhi B, Zmantar T, Hentati H, Bakhrouf A. Cell surface hydrophobicity, biofilm formation, adhesives properties and molecular detection of adhesins genes in S.aureus associated to dental caries. Microbial Pathogenesis 2010; 49(1-2): 14–22.
  2. Thomsen IP, Dumont AL, James DBA, Yoong P, Saville BR, Soper N,  et al. Children with invasive S. aureus disease exhibit a potently neutralizing antibody response to the cytotoxin LukAB. Infection and Immunity 2014; 82(3): 1234–42.
  3. Mandal SM, Ghosh AK, Pati BR Dissemination of antibiotic resistance in methicillin-resistant Staphylococcus aureus and vancomycin-resistant S. aureus strains isolated from hospital effluents. American Journal of Infection Control 2015; 43(12): 87–88.
  4. Brown AF, Leech JM, Rogers TR, McLoughlin RM. Staphylococcus aureus colonization: modulation of host immune response and impact on human vaccine design. Frontiers in Immunology 2014; 4: 507.
  5. Berthelot P, Grattard F, Cazorla C, Passot JP, Fayard JP, Meley R, et al. Is nasal carriage of Staphylococcus aureus the main acquisition pathway for surgical-site infection in orthopedic surgery? European Journal of Clinical Microbiology and Infectious Diseases. 2010; 29: 373-382.
  6. Figueiredo AMS, Ferreira Antunes F, Ossaille Beltrame C, Farrel Côrtes M. The role of biofilms in persistent infections and factors involved in ica-independent biofilm development and gene regulation in Staphylococcus aureus. Critical Reviews in Microbiology 2017; 43: 602–620.
  7. Flemming HC, Wingender J, Szewzyk U, Steinberg P, Rice SA, Kjelleberg S. Biofilms: an emergent form of bacterial life. Nature Reviews Microbiology 2016; 14(9): 563- 75.
  8. Olsen I. Biofilm-specific antibiotic tolerance and resistance. European Journal of Clinical Microbiology and Infectious Diseases 2015; 34: 877–886.
  9. Jamal M, Ahmad W, Andleeb S, Jalil F, Imran M, Asif Nawaz M, et al. Bacterial biofilm and associated infections. Journal of the Chinese Medical Association 2018; 81(1): 7-11.
  10. Gotz F. Staphylococcus and biofilms. Molecular Microbiology 2002;43(6):1367–78.
  11. Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms. Lancet 2001; 358(9276): 135-8.
  12. Hamilton MA. Testing antimicrobials against biofilm bacteria. Journal of AOAC international 2002; 85(2): 479-85.
  13. Le KY, Park MD, Otto M. Immune evasion mechanisms of Staphylococcus epidermidis biofilm infection. Frontiers in Microbiology 2018; 9:359.
  14. Vestby LK, Grønseth T, Simm R, Nesse LL. Bacterial biofilm and its role in the pathogenesis of disease. Antibiotics 2020: 9(2): 59.
  15. Arciola CR, Campoccia D, Montanaro L. Implant infections: adhesion, biofilm formation and immune evasion. Nature Reviews Microbiology 2018; 16(7): 397–409.
  16. Darouiche RO. Device-associated infections: a macro problem that starts with micro adherence. Clinical Infectious Diseases  2001; 33(9):1567–72.
  17. Cherry JD, Yogev R, Harrison GJ, Tan T, Kaplan SL, Steinbach WJ, et al. Infections related to prosthetic or artificial devices. In Feigin, Cherry, Demmier and Kaplan: Textbook of Pediatric Diseases,2013; 7th ed (pp. 1015-1043). Elsevier Saunders.
  18. Namvar AE, Asghari B, Ezzatifar F, Azizi Gh, Rastegar Lari A. Detection of the intercellular adhesion gene cluster (ica) in clinical Staphylococcus aureus isolates. GMS Hygiene and Infection Control 2013; 8(1).  doi: 10.3205/dgkh000203
  19. Arciola CR, Campoccia D, Ravaioli S, Montanaro S. Polysaccharide intercellular adhesin in biofilm: structural and regulatory aspects. Frontiers in Cellular Infection Microbiology 2015; 5:7
  20. Cramton SE, Gerke C, Schnell N F, Nichols W W, Götz F. The intercellular adhesion (ica) locus is present in Staphylococcus aureus and is required for biofilm formation. Infection and Immunity 1999; 67(10): 5427–5433.
  21. McCarthy H, Rudkin JK, Black NS, Gallagher L, O'Neill E, O'Gara1 GP. Methicillin resistance and the biofilm phenotype in Staphylococcus aureus. Frontiers in Cellular and Infection Microbiology 2015; 5: 1. 
  22. Archer NK, Mazaitis MJ, Costerton JW,  Leid JG, Powers ME, Shirtliff ME. Staphylococcus aureus biofilms. Virulence 2011; 2(5): 445–59.
  23. Mathur T, Singha S, Khan S, Upadhyay DJ, Fatma T, Rattan A. Detection of biofilm formation among the clinical isolates of staphylococci: An evaluation of three different screening methods. Indian Journal of Medical Microbiology 2006; 24(1): 25–9.
  24. Zalipour M, Sedigh Ebrahim-Saraieet H, Sarvaria J. Detection of biofilm production capability and icaA/D genes among staphylococci isolates from Shiraz, Iran, Jundishapur Journal of Microbiology 2016; 9(12): 41431.
  25. Mahon C, Lehmanl D. Textbook of diagnostic microbiology-E-Book. Maryland Heights: Elsevier Health Sciences; 2014. Hardcover ISBN: 9780323613170. eBook ISBN: 9780323482127
  26. CLSI Performance Standards for antimicrobial susceptibility testing, 29th edition. CLSI supplement M100. Wayne, PA: Clinical and Laboratory standard Institute; 2019. ISBN Number: 978-1-68440-033-1
  27. Choi SM, Kim SH, Kim HJ, Lee DG, Choi JH, Yoo JH, et al. Multiplex PCR for the Detection of Genes Encoding Aminoglycoside Modifying Enzymes and Methicillin Resistance among Staphylococcus Species. Journal of Korean Medical Science 2003; 18(5): 631-6.
  28. Arciola CR, Campoccia D, Montanaro L. Detection of biofilm-forming strains of Staphylococcus epidermidis and S. aureus. Expert Review of Molecular Diagnostic 2002; 2(5): 478–484.
  29. Halim RMA, Kassem NN, Mahmoud BS. Detection of biofilm producing staphylococci among different clinical isolates and its relation to methicillin susceptibility. Open Access Macedonian Journal of Medical Sciences 2018; 6(8): 1335- 1341.
  30. Christensen GD, Simpson WA, Younger JJ, Baddour LM, Barrett FF, Melton DM, et al. Adherence of coagulase-negative Staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. Journal of Clinical Microbiology 1985; 22(6): 996–1006.
  31. Kadurugamuwa JL, Sin L, Albert E, Yu J, Francis K, DeBoer M, et al. Direct continuous method for monitoring biofilm infection in a mouse model. Infection and Immunity 2003; 71(2): 882–890.
  32. Gad GFM, El-Feky MA, El-Rehewy MS, Amin Hassan M, Abolella H, Mahmoud Abd El-Baky R. Detection of icaA, icaD genes and biofilm production by Staphylococcus aureus and Staphylococcus epidermidis isolated from urinary tract catheterized patients. The Journal of Infection in Developing Countries 2009; 3: 342-51.
  33. Foster TJ. Immune evasion by staphylococci. Nature Reviews Microbiology 2005; 3(12): 948–58.
  34. Edwards AM, Bowden MG, Brown EL, Laabei M, Massey RC. Staphylococcus aureus extracellular adherence protein triggers TNF alpha release, promoting attachment to endothelial cells via protein a. PLoS One 2012; 7(8): 43046.
  35. Tan X, Qin N, Wu Ch, Sheng J, Yang R, Zheng B, et al. Transcriptome analysis of the biofilm formed by methicillin susceptible Staphylococcus aureus. Scientific Reports 2015; 5: 11997.
  36. Piechota M, Kot B, Frankowska-Maciejewska A, Grużewska A, Woźniak-Kosek A. Biofilm formation by methicillin-resistant and methicillin-sensitive Staphylococcus aureus strains from hospitalized patients in Poland. BioMed Research International 2018 : 4657396.
  37. Achek R, Hotzel H, Nabi I, Kechida S, Mami D, Didouh N, et al. Phenotypic and molecular detection of biofilm formation in staphylococcus aureus isolated from different sources in Algeria. Pathogens 2020; 9: 153.
  38. Kot B, Sytykiewicz H, Sprawka I. Expression of the biofilm-associated genes in methicillin-resistant Staphylococcus aureus in biofilm and planktonic conditions. International Journal of Molecular Science 2018; 19(11): 3487.
  39. Yazdani R, Oshaghi M, Havayi A, Pishva E, Salehi R, Sadeghizadeh M, et al. Detection of icaAD gene and biofilm formation in Staphylococcus aureus isolates from wound infections. Iranian Journal of Public Health 2006; 35(2): 25-28
  40. Nguyen HTT, Nguyen ThH, Otto M. The staphylococcal exopolysaccharide PIA - Biosynthesis and role in biofilm formation, colonization, and infection. Computational and Structural Biotechnology Journal 2020; 18: 3324–3334.
  41. Ghaderi H, Malekabad E Sh, Vahidi M, Dadashi AR. Evaluation of genotypic and phenotypic biofilm formation by Staphylococcus aureus isolated from clinical samples and their association with antimicrobial resistance. Iranian Journal of Medical Microbiology 2020; 14(5) :441-459.
  42. Arciola CR, Baldassarri L, Montanaroet L. Presence of icaA and icaD genes and slime production in a collection of staphylococcal strains from catheter-associated infections. Journal of Clinical Microbiology 2001; 39(6): 2151-6.
  43. Rohde H, Knobloch JK, Horstkotte MA, Macket D. Correlation of Staphylococcus aureus icaADBC genotype and biofilm expression phenotype. Journal of Clinical Microbiology 2001; 39(12): 4595–4596.
  44. Diemond-Hern´andez B, Solórzano-Santos F, Leaños-Miranda B, Peregrino-Bejarano L, Miranda-Novales G. Production of icaADBC-encoded polysaccharide intercellular adhesin and therapeutic failure in pediatric patients with staphylococcal device-related infections, BMC Infectious Diseases 2010; 15(10): 68.
  45. Bazari PAM, Honarmand Jahromy S, Zare Karizi Sh. Phenotypic and genotypic characterization of biofilm formation among Staphylococcus aureus isolates from clinical specimens. an Atomic Force Microscopic (AFM) study. Microbial Pathogenesis 2017; 110: 533–539.
  46. Grinholc M, Wegrzyn G, Kurlenda J. Evaluation of biofilm production and prevalence of the icaD gene in methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains isolated from patients with nosocomial infections and carriers. FEMS Immunology and Medical Microbiology 2007; 50(3): 375-379.
  47. Bimanand L, Taherikalani M, Azizi Jalilian F, Sadeghifard N, Ghafourian S, Mahdavi Z, et al. Association between biofilm production, adhesion genes and drugs resistance in different SCCmec types of methicillin resistant Staphylococcus aureus strains isolated from several major hospitals of Iran. Iranian Journal of Basic Medical Science 2018; 21(4): 400–403. 
  48. Mohamadian M, Sadeghifard N, Ghafourian S, Pakzad I. Biofilm formation in Methicillin Resistant Staphylococcus aureus. Gene Reports 2020; 21: 100923. 
  49. Azmi, K, Qrei W, Abdeen Z. Screening of genes encoding adhesion factors and biofilm production in methicillin resistant strains of Staphylococcus aureus isolated from Palestinian patients  BMC Genomics 2019; 20: 578.
  50. John NP, Murugan S. Biofilm Formation by Methicillin Resistant Staphylococcus aureus and their Antibiotic Susceptibility Pattern: An in vitro Study. Current Research in Bacteriology 2014; 7: 1–11.
  51. Hassan A, Usman J, Kaleem F, Omair M, Khalid A, Iqbal M. Evaluation of different detection methods of biofilm formation in the clinical isolates. Brazilian Journal of Infectious Diseases 2011; 15: 303-311.
  52. Oliveira A, Cunha MDL. Comparison of methods for the detection of biofilm production in coagulase-negative staphylococci. BMC Research Notes. 2010; 3: 1-8.
  53. Knobloch JK, Horstkotte MA, Rohde H, Mack D. Evaluation of different detection methods of biofilm formation in Staphylococcus aureus. Medical Microbiology and Immunology. 2002; 191(2): 101-6.
  54. Fitzpatrick F, Humphreys H, O'Gara JP. The genetics of staphylococcal biofilm formation–will a greater understanding of pathogenesis lead to better management of device-related infection? Clinical Microbiology and Infection. 2005; 11(12): 967–73.
  55. Manandhar S, Singh A, Varma A, Pandey Sh, Shrivastava N. Evaluation of methods to detect in vitro biofilm formation by staphylococcal clinical isolates. BMC Reserch Notes. 2018; 11(1): 714.
  56. Saha R, et al. Detection of biofilm formation in urinary isolates: need of the hour. Journal of Research in Biology 2004; 4(1):1174- 1181.
  57. Taj, Y, Essa F, Aziz F, Kazmi ShU. Study on biofilm-forming properties of clinical isolates of Staphylococcus aureus.The Journal of Infection in Developing Countries 2012; 6(5): 403-409.
  58. Abdel Halim RM, Kassem NN, Mahmoud BS. Detection of Biofilm Producing Staphylococci among Different Clinical Isolates and Its Relation to Methicillin Susceptibility. Open Access Macedonian Journal of Medical Sciences. 2018; 6(8):1335-1341.
  59. Johannes KM, Horstkotte MA, Rohde H, Mack D. Evaluation of different methods of biofilm formation in Staphylococcus aureus. Medical Microbiology and Immunology 2002; 191:101-106.
  60. Arciola CR, Campoccia D, Baldassarri L, Donati ME, Pirini V, Gamberini S, Montanaro L. Detection of biofilm formation in Staphylococcus epidermidis from implant infections Comparison of a PCR-method that recognizes the presence of ica genes with two classic phenotypic methods. Journal of Biomedical Material Research. 2006;76(2):425–30.
  61. O’Gara JP. ica and beyond: biofilm mechanisms and regulation in Staphylococcus epidermidis and Staphylococcus aureus. FEMS Microbiology Letters 2007; 270(2): 179–88.
  62. Nasr RA, AbuShady HM, Hussein HS. Biofilm formation and presence of icaAD gene in clinical isolates of staphylococci. Egyptian Journal of Medical human Genetics 2012; 13(3): 269-274.
  63. Moormeier DE, Bose JL, Horswill AR, Bayles KW . Temporal and stochastic control of Staphylococcus aureus biofilm development. American Society for Microbiology, mBio 2014; 5(5): 01341-14.